We propose to greatly advance the signal detection limits of positron emission tomography (PET) by developing a next-generation pre-clinical PET system capable of substantial improvements in visualization and quantification of cellular and molecular signatures of disease. We will build upon advances made in previous work that explored and developed an innovative concept for a 3-D position sensitive photon scintillation detector technology for small animal PET. The proposed project will first greatly improve upon this promising detector technology, substantially (not incrementally) advancing its performance, while also making it even more practical to implement. We will then translate that advanced technology into a small prototype of a box- shaped, small animal PET system with adjustable FOV that we will build from a novel multi-layer detector module. In those detector layers, we will incorporate scintillation crystal arrays with 0.5 mm pixels in order to substantially advance the spatial resolution of small animal PET. This goal is facilitated by the new scintillation detection concept, where the scintillation light collection aspect ratio in each crystal element is very high, even for 0.5 mm width elements. The 0.5 mm resolution goal in reconstructed images is also facilitated by the significantly improved 511 keV photon sensitivity enabled by the box-shaped system design, for reasons that we will clarify in this application. The scintillation crystal arrays are coupled to novel, extremely thin, high gain position sensitive photodetectors arranged in an innovative edge-on configuration that enables directly measured 511 keV photon interaction depth (DOI) within any crystal, and promotes >90% scintillation light collection efficiency, independent of DOI. The resulting robust, non-varying light signal facilitates superior photon energy and temporal resolutions, which, together with 0.5 mm intrinsic spatial resolution, help to enhance PET signal detection and quantification in the presence of background activity. In this detector module's edge-on, layered arrangement, incoming photons traverse a minimum of ~2 cm thick crystal with a crystal packing fraction of 70% in order to promote high 511 keV photon detection efficiency, while the 0.5 mm DOI resolution helps to preserve spatial resolution uniformity throughout the sensitive volume of the resulting PET system. In addition this detector configuration is able to localize individual 511 keV photon interactions occurring in distinct crystal array layers. This is an unusual capability for a PET detector, which we refer to as 3-D positioning. This capability is important for achieving the desired 0.5 mm reconstructed resolution since incoming photons will often interact in multiple crystal elements of the ultra-high resolution detectors. If successful, the proposed 0.5 mm resolution, high sensitivity, 3-D positioning detectors, in conjunction with new event processing algorithms our group is investigating, enable substantial improvements in resolution, contrast, and reconstructed image signal-to-noise ratio. Impact: If successful, this research will advance the ability of PET to detect, visualize and quantify low concentrations of PET tracer accumulating in cells of interest, thus increasing signal detection capabilities for applications in translational cardiovascular, neurological, and cancer research. PUBLIC HEALTH RELEVANCE: We propose to advance the molecular signal detection limits of positron emission tomography (PET) by improving upon a detector technology we have developed under a previous grant and translating the concept into a next-generation high performance pre-clinical PET system. If successful, this work will yield a log order improvement for non-invasively visualizing and quantifying low abundance molecular targets within tissues of live animal subjects. Although there are many applications where this ultra-low signal sensitivity is needed, this advance is especially important in tracking and quantifying the distribution and proliferation of a small population of cells, a capability that is very much needed to guide the development of new cell-based therapies on the horizon.